LOCUS Exported 7004 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Intersectional viral expression of bREACHes-EYFP in cells expressing Cre AND NOT Flp. ACCESSION . VERSION . KEYWORDS pAAV-nEF-Con/Foff 2.0-bREACHes-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7004) AUTHORS Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M, Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K TITLE Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals. JOURNAL Neuron. 2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 10.1016/j.neuron.2020.06.003. PUBMED 32574559 REFERENCE 2 (bases 1 to 7004) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7004 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 177..388 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 401..669 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" protein_bind 697..730 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" regulatory 823..832 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 1309..1342 /label=loxN /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACCT). loxN sites are compatible with each other, but incompatible with loxP or lox2272 sites." protein_bind 1393..1426 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." primer_bind 1539..1560 /label=EGFP-N /note="EGFP, reverse primer" protein_bind complement(2230..2263) /label=loxN /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACCT). loxN sites are compatible with each other, but incompatible with loxP or lox2272 sites." protein_bind complement(2314..2347) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." primer_bind complement(2569..2588) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 2728..2979 /codon_start=1 /product="C-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VC155 /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK" primer_bind 2916..2937 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(2989..3022) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" misc_feature 3131..3719 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3184..3204) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3602..3613) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3751..4227 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3950..3969) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4267..4407 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4267..4396 /label=AAV2 ITR rep_origin 4482..4937 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4569..4588) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4779..4800 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4954..4973) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5073..5095 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5133..5151) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5219..5323 /gene="bla" /label=AmpR promoter CDS 5324..6184 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5542..5561) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6355..6943 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6844..6863 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc 181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg 241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc 421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc 481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc 541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac 601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg 661 ttacagatcc aagctgtgac cggcgcctac ggatccgaag ttcctattct ctagaaagta 721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta 781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccgccaccat ggactatggc 841 ggcgctttgt ctgccgtcgg actgttccag accagctaca ccctggagaa caacggcagc 901 gtgatctgca tccccaacaa cggccagtgc ttctgcctgg cctggctgaa gagtaacggc 961 accaacgccg agaagctggc cgccaacatc ctgcagtggg tggtgtttgc gctgagcgtg 1021 gcgtgcctgg gctggtatgc gtatcaggcg tggcgcgcga cctgcggctg ggaagaagtg 1081 tatgtggcgc tgattgaaat gatgaaaagc attattgaag cgtttcatga atttgatagc 1141 ccggcgaccc tgtggctgag cagcggcaac ggcgtggtgt ggatgcgcta tggctcatgg 1201 ctgctgacct gccccgtgat tctgattcat ctgagcaacc tgaccggcct gaaagatgat 1261 tatagcaaac gcaccatggg cctgctggta agtgtcgggg ttgagctcat aacttcgtat 1321 agtatacctt atacgaagtt attctttgca ccattctaaa gaataacagt gataatttct 1381 gggttaaggc aaataacttc gtataggata ctttatacga agttatggta cccctcctgt 1441 actcaccttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc 1501 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag 1561 gatgggcacc accccggtga acagctcctc gcccttgctc accacgttga tgtcgatctg 1621 gtccaggggg atgtactcgc cctcgctggt gatcctgctc ttggcggccg catcttcttc 1681 ttccgccacc agggtttcca cttccatttc ctggcccgca atggtaattt tctgtttttt 1741 gcgaatatcg ccatacagca gaatatgttc atgaattttc acgcgcagat agttgcccag 1801 cacgccccac atgttcttcg caatcagatc cagaatgcta tggccaatcg cgctgccata 1861 cgggctaata tggccgaagc cctcggggcc cagcaggaac agcacgggga acatgcccca 1921 gctcacgaag aacagccagg ccatggctct caccagctgt ctgcacaggc ctttcggcac 1981 ggtatgaaac gcttcaatat acactttggc cgcatgaaaa taggtataca tgccatagct 2041 caggctaatc agaaaaaaca gaattttggt ccagccggtg cacatcgcgc tggtcgcgcc 2101 ccacacaatg cagcccacgt cgctcaccta aacacatgag aaatagagtc ataagcacat 2161 gggtcacata caggagatat gtatataaca ttaatacaat ttataaaaaa aagggggggc 2221 acaactagta taacttcgta taaggtatac tatacgaagt tattaagagg tttcatattg 2281 ctaatagcag ctacaatcca gctaccattc tgcataactt cgtataaagt atcctatacg 2341 aagttatgtc gactggagag tggccagccc ttctcatgtt cagagaacat ggttaactgg 2401 ttaagtcatg tcgtcccaca gctgcccgtg ccctggccca ccctcgtgac caccttcggc 2461 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag 2521 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 2581 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 2641 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 2701 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 2761 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 2821 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc 2881 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 2941 gccgccggga tcactctcgg catggacgag ctgtacaagt aagctagcga agttcctata 3001 ctttctagag aataggaact tctaagcaga caatcttgta atacatcagc agctagtaga 3061 ttgctccagc tagaagttcc tatacctttt gaagaatagg aacttcgaat tcgatatcaa 3121 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc 3541 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca 3721 tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg 3781 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta 3841 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg 3901 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa 3961 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa 4021 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc 4081 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc 4141 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg 4201 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc 4261 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 4321 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 4381 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 4441 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 4501 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt 4561 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 4621 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 4681 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 4741 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 4801 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc 4861 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 4921 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 4981 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 5041 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 5101 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 5161 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 5221 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 5281 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 5341 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 5401 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 5461 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 5521 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 5581 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 5641 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 5701 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 5761 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 5821 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 5881 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5941 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 6001 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 6061 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 6121 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 6181 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 6241 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 6301 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 6361 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 6421 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 6481 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 6541 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6601 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6661 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6721 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6781 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6841 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6901 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6961 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt //